Alcohol dehydrogenase

Alcohol dehydrogenase (PDB id 1htb), ADH, is an 80kDa enzyme that catalyzes the 4th step in the metabolism of fructose before glycolysis. In the 4th step, glyceraldehyde is converted to the glycolytic intermediate DHAP by the NADH-dependent, ADH catalyzed reduction to glycerol. ADH catalyzes the oxidation of primary and secondary alcohols to their corresponding aldehydes and ketones through a mechanism that involves the removal of a hydrogen. For detailed discussion of horse liver alcohol dehydrogenase see Horse Liver Alcohol Dehydrogenase.

Structure
The initial scene (Domains of ADH ) shows an overview of the molecule, allowing for a general look at the tertiary structure of alcohol dehydrogenase (it is complexed with Cl, Pyz, NAD, and Zn). A second scene (Closer Look at Subunit ) shows a close view of the ligand within each subunit. Labels have been placed on NAD, CL, and Zn to clearly establish the structure.

Within alcohol dehydrogenase, the active site of alcohol dehydrogenase has three important residues, Phe 93, Leu 57, and Leu 116. These three residues work together to bind to the alcohol substrate.

Zn plays an important role in the catalysis. It funtions by electrostatically stabilizing the oxygen in alcohol during the reaction, which causes the alcohol to be more acidic. At the Zinc Binding Site, Zinc coordinates with Cys 146, Cys 174, and His 67.

NAD functions as a cosubstrate in the dehydration. NAD binds to numerous residues in a series of beta-alpha-beta folds. NAD Binding Region shows the domain where NAD binds, and many of the residues with which it interacts are selected.

Alcohol dehydrogenase exists as a dimer with a zinc molecule complexed in each of the subunits. It has a SCOP catagory of an alpha and beta protein. At the N-terminal, there is a domain that is all beta; however, the C-Terminal domain is alpha and beta, so the catagory is alpha and beta. The C-Terminal core has 3 layers of alpha/beta/alpha and parallel beta sheets of 6 strands.

Reaction and Mechanism
In the oxidation mechanism, ADH is momentarily associated with nicontinamide adenine dinucleotide (NAD+), which functions as a cosubstrate. In its reaction, alcohol dehydrogenase uses zinc and NAD to facilitate the reaction. The function of zinc is to position the –OH group on the ethanol in a conformation that allows for the oxidation to occur. NAD then acts as a cosubstrate and performs the oxidation.

The of alcohol dehydrogenase reaction is as follows: CH3CH2OH + NAD+ -> CH3COH (acetaldehyde) + NADH + H+ (Note: The reaction is actually reversible although the arrow does not show it) The step-wise reduction mechanism for ADH is shown on the left. In the mechanism, His 51 is deprotonated and activated by a base catalyst. This allows histidine to accept a proton from NAD, which also draws a proton Thr 48. As a result of the proton transfer, the Thr is prepared to accept a proton from the alcohol substrate. While Thr accepts the proton, there is also a hydride transfer to NAD. The whole process can be summarized as the oxidation of an alcohol to an aldehyde in concert with the transfer of a hydride to NAD.

The Mechanism for alcohol dehydrogenase follows an random bisubstrate mechanism. In the mechanism, the NAD+ and alcohol bind to the enzyme, so that the enzyme is now attached to the two subtrates. While attached, the hydrogen is formally transferred from the alcohol to NAD, resulting in the products NADH and a ketone or aldehyde. The two products are then released, and the enzyme has catalyzed the reaction.

Kinetics
The alcohol dehydrogenase catalyzed aldehyde-NADH reaction show kinetics consistent with a random-order mechanism, and the rate-limiting step is the dissociation of the product enzyme-NAD+ complex. Alcohol dehydrogenase is more effective for smaller alcohol substrates, and it becomes less effective as substrate size increases. It is also more effective for primary than secondary alcohols. In a study where ADH was immobilized in tresyl-chloride-activate agarose, it was shown that the Michaelis-Menten model could not take into consideration all the constraints induced by the immobilization on the enzyme properties but that the Theorell-Chance model was more appropriate.

Regulation
Substrate size is a regulator, where larger substrates inhibit alcohol dehydrogenase. Further, alcohol dehydrogenase is somewhat inhibited if the substrate is a secondary alcohol, as opposed to a primary alcohol. Pyrazoles have also been shown to be inhibitors of ADH. Other inhibitors include heavy metals, thiourea, purine and pyrimidine derivatives, and both chloroethanol and flouroethanol. Activators include sulfhydryl activating reagents, mercaptoethanol, dithiothreitol, and cysteine.

Tetrameric alcohol dehydrogenases


The NADP+-dependent alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii (TbADH), the mesophilic bacterium Clostridium beijerinckii (CbADH), and the protozoan parasite Entamoeba histolytica (EhADH1) are homotetrameric (monomers are colored in different colors) secondary alcohol dehydrogenases. Each monomer of these alcohol dehydrogenases consists of two domains: the cofactor-binding domain  (residues 154−294 for TbADH) and the catalytic domain (residues 1−153 and 295−351 for TbADH ; contains Zn2+ at the active site) separated by a deep cleft. Although, all these three ADHs revealed a high degree of sequence conservation (62-75% identity), them significantly differ in thermostability. The cofactor-binding domains (residues 153−295) of TbADH, CbADH, and EhADH1 were mutually exchanged and 3 corresponding chimeras were constructed. The cofactor-binding domain of thermophilic TbADH was replaced with the cofactor-binding domain of its mesophilic counterpart CbADH (chimera Χ21(TCT), 3fsr). This domain replacement significantly destabilized the parent thermophilic enzyme (ΔT1/2 = −18 °C). But the reverse exchange in CbADH (chimera Χ22(CTC), 3fpl), had little effect on the thermal stability of the parent mesophilic protein. The exchange of the cofactor-binding domain of TbADH with the homologous domain of EhADH1 (chimera Χ23(TET), 3fpc) substantially reduced the thermal stability of the thermophilic ADH (ΔT1/2 = −51 °C) and interfered the oligomerization of the enzyme.

The double mutant of the chimera Χ21(TCT) (cofactor-binding domain of thermophilic TbADH replaced by that of mesophilic CbADH) Q165E/S254K-X21(TCT) (3ftn) was constructed by site-directed mutagenesis. In both TbADH and CbADH, Lys257 and Asp237 form an intrasubunit ion pair, in TbADH, Asp237 is also involved in an ion pair bridge with Arg304 of the adjacent monomer. In addition, Arg304 forms intersubunit salt bridge with Glu165 of the first monomer. Therefore, a four-member ion pair network involving Lys257, Asp237, and Glu165 of one monomer and Arg304 of the adjacent one is present in TbADH (the names of monomers are in brackets). However in mesophilic CbADH (and, therefore, in the chimera Χ21(TCT), 3fsr) the Gln is situated in position 165 (instead Glu of TbADH) and Met in position 304 (instead Arg of TbADH), so, such an ion pair network does not exist. In the double mutant Q165E/S254K-X21(TCT) reverse mutation Q165E reconstructs this network (as in parent thermophilic TbADH) that led to significant enhancement of the thermal stability of CbADH (ΔT1/260 min = 5.4 °C). Chimera X21(TCT) (3fsr) is colored magenta and the double mutant Q165E/S254K-X21(TCT) cyan (3ftn). In chimera X21(TCT), position 254 is occupied by Ser (due to sequence of exchanged domain). The replacement of Ser254 of CbADH with Lys significantly enhances the stability of the enzyme, due to the formation of <scene name='3fsr/Al/3'>intrasubunit Lys254 and Glu280 ion pair. However, this replacing of Ser254 by Lys had a negligible effect on the thermal stability, in contrast to mutation Q165E mentioned above.

The <scene name='3fsr/Al1/2'>comparison of overall Cα backbone of all these chimeras (rmsd 0.45-0.65 Å) with those of the parent enzymes, did not reveal significant structural changes. So, the differences in the thermal stability of the chimeras and the parent enzymes could be caused by relatively small specific changes located at the important points of the NADP+-dependent alcohol dehydrogenases. For example see Cα superposition for the <font color='red'>X23(TET) chimera (red) (3fpc) and its parent ADHs (<font color='blue'>TbADH, colored blue (1ped), and <font color='lime'>EhADH1, colored lime (1y9a). The RMSDs of the TbADH−EhADH1, TbADH−Χ23(TET), and EhADH1−Χ23(TET) were 0.68, 0.56, and 0.48 Å, respectively.

The 3D structure of CbADH with the substitution Q100P (<scene name='2b83/Tet/3'>tetramer ) was solved at 2.25 Å resolution (2b83). The <scene name='2b83/Mut/1'>substitution of Gln100 with Pro did not cause significant structural changes in the protein structure. The residues of the <font color='lime'>wildtype protein are colored lime and the residues of the <font color='cyan'>mutant one in cyan. Only 2 H-bonds were lost, one between Oε1 of Gln100 and the main chain N of Gly297, and the second between Nε2 of Gln100 and the main chain carbonyl O of Gly297. The mutation caused that an additional CH2 group (Cδ of Pro100) is surrounded by nonpolar residues: Pro88 (3.8 Å), Trp90 (3.5 Å), and Val95 (4 Å). These residues (P100, P88, W90, and V95) are situated on a protruding lobe of the protein. An additional 11 aliphatic and aromatic carbon atoms are situated within the distance of 6 Å from Cδ of Pro100 (two methyl groups of Val95; three carbon atoms of the Trp90 indole group; Cβ and Cγ methylene groups of Pro100; Cβ and Cγ of Gln101, and two carbons of the Phe99 phenyl ring).

Ribbon diagram of the EhADH1 <scene name='2oui/Tet/1'>tetramer (2oui). Proline residues (ball representation) are colored <font color='orange'>orange (Pro275) (which is important for thermal stabilization) and <font color='cyan'>cyan (Pro100). <scene name='2oui/Tet/5'>Superposition of the structures of the <font color='lime'>wild-type apo-EhADH1 (colored lime, 1y9a) and the <font color='orange'>apo D275P-EhADH1 mutant (colored orange) (2oui). <font color='red'>Pro275 and Asp275 are labeled red. Residues within a distance of 4 Å from the mutation are shown (names of monomers are in brackets). Replacing <scene name='2oui/Tet/8'>Asp275 with <scene name='2oui/Tet/7'>Pro significantly enhanced the thermal stability of EhADH1: ΔT1/260min = +9.3°C, ΔT1/2CD = +10°C. The reverse mutation in the thermophilic <scene name='Tetrameric_alcohol_dehydrogenases/Mut/3'>TbADH (1ykf; <font color='magenta'>colored magenta ) - substitution of wt TbADH Pro275 with <scene name='Tetrameric_alcohol_dehydrogenases/Mut/2'>Asp (2nvb; <font color='cyan'>colored cyan ) reduced the thermal stability of the enzyme: ΔT1/260min = -13.8°C, ΔT1/2CD = -18.8°C. Nitrogen and oxygen atoms are colored in CPK colors. <font color='red'>Pro275 and Asp275 are labeled red (names of monomers are in brackets). These findings indicate that a single proline mutation is responsible for the significant differences in the thermal stability of ADHs, and show the importance of prolines in the protein stability. It was also shown that substitution by proline at the important positions could significantly stabilize the protein.

</StructureSection>

Additional Resources
For additional information, see: Carbohydrate Metabolism

3D Structures of Alcohol dehydrogenase
Update June 2011

ADH I
3jv7 – RrADH I – Rhodococcus rubber

2vna - hADH I catalytic domain - human

2hcy – yADH I – yeast

ADH I binary complex

1u3t – hADH I α chain + inhibitor

1hsz, 1hdz, 3hud - hADH I β chain + NAD

1u3w - hADH I γ chain + inhibitor

1ht0 - hADH I γ chain (mutant) + NAD

ADH I ternary complex

2xaa – RrADH I + NAD + alcohol

3fx4 – pADH I + NADP + inhibitor – pig

2w98, 2w4q – hADH I catalytic domain + NADP + inhibitor

1hso - hADH I α chain + NAD + pyrazole derivative

1hdx - hADH I β chain + NAD + alcohol

1u3u, 1u3v - hADH I β chain + inhibitor

1deh, 1hdy - hADH I β chain + NAD + pyrazole derivative

1htb - hADH I β3 chain + NAD + pyrazole derivative

ADH II
3owo – ZmADH II iron-dependent – Zymomonas mobilis

ADH II binary complex

3ox4 - ZmADH II iron-dependent + NAD

3cos - hADH II + NAD + Zn

1e3e – mADH II + NADH – mouse

1e3l - mADH II (mutant) + NADH

1e3i - mADH II + NADH + inhibitor

ADH III

1m6h, 1m6w, 1teh - hADH III χ chain

2fze - hADH III χ chain + ADP-ribose

2fzw - hADH III χ chain + NAD

1mc5 – hADH III χ chain + glutathione + NADH

1ma0 - hADH III χ chain + dodecanoic acid + NAD

3qj5 - hADH III χ chain + inhibitor + NAD

ADH IV
1ye3, 8adh, 5adh - hoADH IV e chain – horse

1qlj - hoADH IV e chain (mutant)

3iv7 – ADH IV – Corynebacterium glutamicum

ADH IV binary complex

2jhf, 2jhg, 1het, 1heu, 1hf3, 1ee2, 2oxi, 2ohx, 6adh - hoADH IV e chain + NAD

1adb, 1adc, 1adf, 1adg, 7adh - hoADH IV e chain + NAD derivative

1mgo, 1ju9, 1qlh, 1a72 - hoADH IV e chain (mutant) + NAD

1d1s, 1agn – hADH IV σ chain + NAD

1d1t - hADH IV σ chain (mutant) + NAD

ADH IV ternary complex

3oq6, 1qv6, 1qv7, 1a71, 1axe, 1axg – hoADH IV e chain (mutant) + NAD + alcohol

1p1r, 1ldy, 1lde - hoADH IV e chain + NADH + formamide derivative

1n92 - hoADH IV e chain + NAD + pyrazole derivative

1bto, 3bto - hoADH IV e chain + NADH + butylthiolane derivative

1n8k - hoADH IV e chain (mutant) + NAD + pyrazole

1mg0, 1hld - hoADH IV e chain + NAD + alcohol

ADH
1a4u – SlADH – Scaptodrosophila lebanonensis

3my7 – ADH ACDH domain – Vibrio parahaemolyticus

3meq – ADH – Brucella suis

3l4p – ADH – Desulfovibrio gigas

1jvb - SsADH – Sulfolobus solfataricus

3i4c, 1nto, 1nvg – SsADH (mutant)

3goh – ADH – Shewanella oneidensis

3gaz – ADH residues 2-334 – Novosphingobium aromaticivorans

2eih – ADH – Thermus thermophilus

1rjw – ADH – Geobacillus stearothermophilus

1vj0, 1vhd – TmADH -Thermotoga maritima

2eer – ADH – Sulfolobus tokodaii

ADH binary complex

3l77 – ADH short-chain + NADP – Thermococcus sibiricus

1h2b – ADH + NAD – Aeropyrum pernix

1f8f – Benzyl-ADH + NAD – Acinetobacter calcoaceticus

1o2d - TmADH + NADP

1b16, 1b14, 1b15 - SlADH + NAD derivative

1cdo – ADH + NAD - cod

1rhc – ADH F420-dependent +F420-acetone – Methanoculleus thermophilus

1agn – hADH (sigma) +NAD

ADH ternary complex

1mg5 – ADH + NADH + acetate – Drosophila melanogaster

1r37 – SsADH + NAD + alcohol

1sby – SlADH + NAD + alcohol

1b2l - SlADH + NAD + cyclohexanone

1llu - ADH + NAD + alcohol – Pseudomonas aeruginosa

3cv7 – pADH + NAD + NAP

NADP-dependent ADH
1ped - CbADH – Clostridium beijerinckii

2b83, 1jqb – CbADH (mutant)

2nvb - TbADH (mutant) – Thermoanaerobacter brockii

3ftn, 3fpc, 3fpl, 3fsr – ADH chimera

1y9a - EhADH – Entamoeba histolytica

2oui – EhADH (mutant)

1p0c – RpADH8 – Rana perezi

NADP-dependent ADH binary complex

1kev – CbADH + NADPH

1bxz – CbADH catalytic domain + alcohol

1ykf – TbADH + NADP

3h4g – pADH + NADP

1p0f – RpADH + NADP

R-specific ADH
1nxq - LbRADH – Lactobacillus brevis

1zk2, 1zk3 - LbRADH (mutant)

1zjy, 1zjz, 1zk0, 1zk1 – LbRADH (mutant) + NADH + alcohol

1zk4 - LbRADH (mutant) + NADH + acetophenone

Specific alcohol ADH
2cf5, 2cf6 – Cinnamyl-ADH – Arabidopsis thaliana

1piw, 1q1n, 1ps0 – Cinnamyl-yADH

1m2w – Mannitol-ADH – Pseudomonas fluorescens

1w6s – Methanol-ADH – Methylobacterium extorquens

1yqx – Sinapyl-aADH II – aspen

1yqd – Sinapyl-aADH II + NADP

1bdb – Biphenyl dihydrodiol-ADH + NAD - Pseudomonas

Quinohemoprotein ADH
1kv9, 1yiq – PpQADH II + PQQ + heme – Pseudomonas putida

1kb0 - QADH I + PQQ + heme – Comamonas testosteroni

Hydroxyacyl-CoA dehydrogenase
2et6 – HADH – Candida tropicalis

1zcj – rHADH - rat

1e3s – rHADH II + NADH

1gz6 - rHADH II residues 1-319 + NADH

1e3w - rHADH II + NADH + keto butyrate

1e6w - rHADH II + NADH + alcohol

1lsj, 1lso– hHADH (mutant) + NAD